1. Preparation of Small Lipid Vesicles
<ol>
	<li>Prepare a solution of lipids SPE/DOPS/ATTO488-DOPE in chloroform with a mass mixing ratio of 79/20/1.The final concentration of lipids in chloroform is 1 mg/mL, and the final mass is 0.6 mg (typically less than 1 mg). Use disposable round bottom glass vials (round bottom glass tubes, 10 x 75 mm) when preparing the lipid solution without a pre-cleaning step.
	<ol>
		<li>Fill and rinse the glass syringes (25 &#181;L) with chloroform at least 5 times before usage. To fill and rinse the glass syringe at least 5 times, 3-4 mL of chloroform is sufficient. 
		CAUTION: When handling chloroform, use glass syringes and perform all the procedures in a fume hood while wearing appropriate gloves and protective eyewear at all times. Do not use any plastic flasks, syringes, or pipettes when handling chloroform.</li>
	</ol>
	</li>
	<li>Wrap the glass vial containing the lipid mixture with metal foil to protect the contents from ambient light and evaporate the solvent in a rotary evaporator for 3 h to remove the chloroform, with the pressure reaching 7 kPa vacuum at 80 rpm. A dry lipid film is formed at the bottom of the glass vial after evaporation (Figure 2A). 
	NOTE: Scaling up several times and using larger amounts of lipids may require longer rotary evaporation time to ensure full removal of chloroform.</li>
	<li>Slowly add 600 &#181;L of phosphate buffered saline (PBS) buffer solution (pH 7.8, without dilution) on top of the dried lipid film. Following the addition of the buffer, gently add 6 &#181;L of glycerol to protect the sample from complete dehydration during the GUV-MLV formation steps (achieving a final concentration of glycerol of 1 wt%)36. Seal the glass vial using parafilm and cover with metal foil to protect from ambient light. Store in the fridge at 4 &#176;C overnight. 
	NOTE: The PBS buffer solution consists of 0.151 g of Trizma base, 1.592 g of K3PO4, 1.021 g of KH2PO4, 0.062 g of MgSO4 &#183;7H2O, and 0.0465 g of EDTA in 250 mL of purified water. Store the solution at 4 &#176;C, and warm to room temperature prior to usage. The equivalent amount of 10 mM HEPES buffer can be used instead of PBS solution. The HEPES buffer solution is prepared by diluting a 1 M HEPES stock solution with purified water.</li>
	<li>The following day, sonicate the glass vial containing the solution for 1 min using an ultrasonication bath at room temperature. Remove the parafilm and pipette until a visually uniform solution of small lipid vesicles is formed (Figure 2B). Aliquot 30 &#181;L of the obtained small lipid vesicle solution into individual 0.5 mL plastic&#160;tubes. Keep these stock aliquots at -18 &#176;C for a maximum of 6 months. 
	NOTE: If the small lipid vesicle solution is not visually uniform, vortex the solution 4 times for 1-2 s using a vortex mixer at maximum speed.</li>
</ol>
2. Preparation of GUV-MLV Complexes
<ol>
	<li>Thaw to room temperature a plastic&#160;tube containing an aliquot of the frozen suspension of small lipid vesicles. Vortex the tube 4 times for 1-2 s using a vortex mixer at maximum speed.</li>
	<li>Place 5 &#181;L of the small lipid vesicle suspension onto the surface of a glass cover slip (24 x 60 mm) to form a small round droplet. Use a glass cover slip without any pre-cleaning steps.</li>
	<li>Place the glass cover slip in a vacuum desiccator (&#60;100 kPa vacuum) for 20 min.</li>
	<li>Store the dried lipid film at room temperature for 4 min, then slowly pipette 50 &#181;L of 10 mM HEPES buffer on top of the dry lipid film for rehydration. Wait 5 min to pre-form the GUV-MLV complexes (Figure 2C).</li>
	<li>Center a glass cover slip on the microscope stage, then pipette 300 &#181;L of HEPES buffer on it and position the center of the droplet above the objective (Figure 3B).</li>
	<li>Transfer the 50 &#181;L of the pre-formed GUV-MLV solution into the HEPES solution (300 &#181;L). Wait 25 min to allow sparsely formed GUV-MLV complexes to firmly adhere to the surface of the glass cover slip (Figure 2D). 
	NOTE: Occasionally, the GUV-MLV complexes do not form on the glass cover slip and instead lipid patches or only MLVs are observed. This can be explained by accidental shaking of the sample during steps 2.4 or 2.6, or numerous freeze-thaw cycles of the small vesicle suspension. Repeat steps 2.1-2.6 or use freshly prepared lipid stock solution (step 1.1) to obtain the GUV-MLV complexes.</li>
</ol>
3. Micropipette Preparation and Microinjection
<ol>
	<li>Round the edges of the borosilicate glass capillaries by gently placing the capillary ends into the flame of a candle to prevent the micropipette from being broken while attaching it to the micropipette holder.</li>
	<li>Pull at least 3 glass capillaries using an automatic laser puller using the program set: Heat = 400, Fil = 4, Vel = 50, Del = 225, Pul = ; Heat = 400, Fil = 4, Vel = 60, Del = 150, Pul = 120. The program set must be entered with the first value of Pul left blank. Using borosilicate glass capillaries of inner diameter (I.D.) 1.00 mm and outer diameter (O.D.) 0.78 mm results in a micropipette with a tip opening of approximately 0.3 &#181;m in diameter.</li>
	<li>Back-fill each micropipette with 8 &#181;L of 5 mM CaCl2 solution in HEPES buffer (10 mM) using a microloader. Filter the CaCl2 solution using a 0.2-0.5 &#181;m syringe filter prior to usage to avoid clogging of the pipette tip. 
	NOTE: Ensure that the CaCl2 is completely dissolved in the HEPES buffer solution, which is also used for steps 2.4-2.6.</li>
	<li>Connect a micropipette holder to a micromanipulator. Mount the micropipette into the micropipette holder tightly. Connect the injection pump and capillary holder using the supply tube.</li>
	<li>Start the injection pump. Adjust the settings on the injection pump to 20 hPa (injection pressure) and set a 5 hPa compensation pressure, on automatic mode. Pause the injection pump until the micropipette is ready for the microinjection.</li>
	<li>Set the microscope to brightfield mode. Configure it for differential interference contrast (DIC). Use 63X or 100X high NA (1.3) objectives to achieve the optimal membrane edge resolution.</li>
	<li>Use the coarse micromanipulator to position the micropipette above the droplet containing the GUV-MLV complexes. Locate the tip of the micropipette above the objective.</li>
	<li>Focus the microscope on the midpoint of the GUV-MLV complex and then refocus approximately 50 &#181;m above the GUV. Use the coarse mode of the micromanipulator to gently bring the micropipette into focus.</li>
	<li>Switch the micromanipulator to the fine mode. Slowly refocus to the GUV surface while translating the micropipette tip down and keeping the tip in focus. Position the micropipette tip approximately 20 &#181;m from the GUV surface. If the micropipette tip is broken due to accidental contact with the glass cover slip (example in Figure 5B), replace it immediately to avoid unnecessary calcium ion release into the sample bulk solution.</li>
</ol>
4. Formation and Translation of MTPs using the Calcium Ion Source
<ol>
	<li>Set the microscope into fluorescence mode. Set dichroics to excite at 488 nm and detect the emission at 505-550 nm.</li>
	<li>Switch on the injection pump and start the calcium ion injection. Use the fine mode of the micromanipulator, and slowly approach the GUV with the micropipette tip. Position the micropipette tip at a distance of 3 &#181;m from the membrane surface while injecting the CaCl2 solution from the micropipette to allow MTPs to form (Figure 4).</li>
	<li>To translate the MTPs along the GUV surface, slowly move the pipette tip (with a velocity of 0.1-0.3 &#181;m/s) around the GUV surface using the fine micromanipulator (Figure 6). Maintain approximately the same distance (3 &#181;m) between the GUV surface and the micropipette tip while continuing the calcium ion injection. 
	NOTE: At high translation rates of the micropipette tip (above 0.7 &#181;m/s), the MTPs will not migrate in sync with the micropipette. Instead, they will scatter and shorten, while new MTPs will be formed at the new location of the micropipette tip (Figure 7).</li>
	<li>To stop the microinjection, turn off the injection pump and move the micropipette away from the GUV surface.</li>
</ol>

Biomimetic cell systems allow for the study of membrane behavior upon the exposure to external stimuli such as ions, proteins, or nanoparticles. GUVs, being such a model, can respond to alterations in the chemical environment by adjusting their shape, which often involves the formation of tubular structures and invaginations24,41,42,43.
This article offers an approach to generate MTPs in a contactless manner via remodeling of the GUV surface upon localized injection of calcium ions at the GUV surface. The protocol describes the preparation of the GUV-MLV complex, which mimics a cell plasma membrane, as well as how to employ a microinjection technique to generate calcium ion gradients close to the GUV surface to form MTPs. The majority of previous experimental studies that addressed calcium ion-membrane interactions exposed lipid vesicles to bulk calcium ion concentration14,17. Depending on the experimental conditions, such bulk exposure may result in a different membrane response with no tubular protrusions formed25.
The formation of the GUV-MLV complexes is rather straightforward and only requires standard laboratory equipment, such as a rotary evaporator, ultrasonic bath, and vacuum desiccator. Still, there are several critical steps to consider during the vesicle preparation. It is important to ensure that the dehydration of the vesicles is complete (during step 2.3 of the protocol) and a dry circular lipid film containing only a small amount of salt crystals is formed on the surface of the glass cover slip. During the experiment, careful handling of the vesicle solution, using fresh lipid stock solution in chloroform as well as fresh HEPES buffer, is essential for successful preparation of the GUV-MLV complexes. Furthermore, secure attachment of the GUV-MLV complex to the glass cover slip surface is crucial for micromanipulation and microinjection. To confirm adequate adhesion of the GUV-MLV complex, the micropipette (without injection flow) can be used to gently push against the GUV surface. A firmly adhered vesicle will not slide along the surface upon direct physical contact. Because the experiments are performed in an open buffer droplet, which may be interrogated for several hours, evaporation must be taken into consideration. Evaporation from the buffer droplet will change the osmotic conditions, which could affect and destabilize the vesicles. To restore osmotic conditions, periodical addition of pure water to the sample to restore the original volume returns the system to homeostasis.
When modifying the lipid membrane composition, it is crucial that the vesicles are produced in the form of a GUV-MLV complex because the MLV allows for transferring the lipid material to the GUV during the membrane reshaping. Previous studies have shown that replacing the components of the SPE mixture with pure lipids, or adding 5-30% of cholesterol, also allows for GUV-MLV complex formation28,44. The majority of the prepared GUVs are unilamellar45.
Furthermore, when testing other divalent cations, such as magnesium ions, the formation of the MTPs heavily depends on the presence of negatively charged DOPS in the lipid mixture. Without DOPS, the MTPs do not form in the vesicles described in this protocol. Also, monovalent cations, such as potassium and sodium, do not result in the formation of MTPs, even in DOPS-containing vesicles25.
In addition to the critical steps with regard to preparation and manipulation of the GUVs, there are several important factors to consider during the microinjection procedure. The successful microinjection of calcium ions heavily relies on properly functioning glass micropipettes, which are prepared on the day of the experiment. There are several factors that could cause the micropipette to malfunction. A common reason is a clogged tip opening. Small lipid particles, which are byproducts of the vesicle preparation, are dispersed in the solution and tend to adhere to the micropipette tip, thereby generating a clogging. Cleaning the pipette tip is best done by lifting it up from the vesicle solution and placing it back close to the GUV surface. The usage of the blow-out function of the microinjection pump must be avoided because it results in massive injection of calcium ions into the bulk solution. Furthermore, small air bubbles entrapped inside the micropipette prevent proper microinjection, in which case the micropipette should be replaced with a new one. Tip breakage can be significantly minimized by placing the experimental setup on a vibration-dampening table to minimize tip oscillations.
Furthermore, care needs to be taken when choosing an observation scheme, to minimize photobleaching whilst obtaining the best time-resolved images. Wide-field laser-induced fluorescence microscopy was utilized in this protocol because it allows for a relatively high image acquisition rate at an objective limited probe depth. Furthermore, usage of inverted microscopy allows for simultaneous microinjection and observation of the lipid vesicles and MTPs.
One of the main limitations of the presented method is the requirement of extensive manual work and sufficient micromanipulation skills. Because the complexes are formed through a spontaneous swelling process, the size of the GUVs and MLVs cannot be controlled. Additionally, this protocol does not allow for control of the membrane tension of the prepared GUV-MLV complexes, which might be necessary to collect additional details with regard to membrane remodeling. The GUVs are connected to the MLVs with the latter supplying lipid material for the substantial growth of the MTPs to an extent that would be impossible to achieve solely utilizing the membrane available from the GUVs. The MLVs also contribute to lowering any lateral surface tension variations within the GUV-MLV complex44, which would complicate the attempts to control the tension of the vesicle by using micropipette aspiration. This GUV-MLV-based model does provide a lower tension that better mimics the tension regimes found in cellular membrane structures connected to membrane reservoirs, such as membrane folds and invaginations46. At the same time, the micropipette aspiration technique can be successfully applied to control membrane tension in single GUVs. For example, the work by Graber et al. provided details on the formation of membrane tubular invaginations in single GUVs upon binding of calcium ions to the membrane at bulk conditions from varied tension regimes40. Finally, the comparison of the membrane behavior at both local and bulk exposure to calcium requires improved control of the membrane adhesion to the surface, which is beyond the scope of this protocol.
To summarize, the proposed technique allows for contactless membrane remodeling and formation of MTPs upon localized stimulation with calcium ions. Future applications of this method center on the translation from synthetic vesicle systems to native biological membranes, such as cell blebs. The proposed method can be incorporated with other single-cell interrogation schemes, such as patch-clamp or microelectrode amperometry, or combined with localized heating strategies31,47,48. Testing the effect of other ions or molecules is straightforward and involves simply replacing the calcium ions with the molecules of interest. Furthermore, complex synthetic lipid vesicles can be produced through membrane functionalization with transmembrane proteins, which may expand our understanding of the biophysics of cell reshaping and cell membrane dynamics associated with sensing of local chemical gradients. Last but not least, contactless stimulation of the lipid membrane can also be translated to polymeric soft matter systems, offering a basis for a novel contactless manipulation platform.

The role of calcium ions within biological processes, specifically their involvement in signaling, cell division, and membrane fusion, is the focus of many mechanistic studies1. The intracellular cytoplasmic calcium ion concentration is on the order of 100 nM, while the calcium in organelles, such as the endoplasmic reticulum, secretory vesicles, and mitochondria, reach levels up to tens of millimolars in concentration. This creates steep calcium ion concentration gradient orders of magnitude across intracellular membranes2,3,4,5,6,7,8,9. The extracellular calcium ion level is around 2 mM and hence, the variations of calcium ion concentration occur at both the extracellular and intracellular levels. Furthermore, recent studies provide evidence that intracellular calcium ion signaling events and neuronal activity can occur under the conditions of local fluctuations of extracellular calcium ion concentrations, indicating the importance of synchronized intra- and extracellular calcium ion variations10.
Aiming to understand the interplay between calcium ions and biological membranes, a synthetic approach in which native cell membranes are replaced with lipid bilayer vesicles has been successfully implemented. Exposing vesicles to calcium ion solutions leads to changes in lipid head groups and hydrocarbon chain packing, increased membrane tension, and vesicle aggregation, as well as the segregation of lipids and membrane phase transition11,12,13,14,15,16. The properties of the lipid membranes upon exposure to calcium ions have been investigated using such experimental techniques as X-ray, 1H-NMR, and spectroscopic or thermodynamic studies11,16,17,18. In these studies, the membrane composition is often tuned to resemble native cell membranes and contains such physiological lipids as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). PS is especially important in artificial vesicle preparation because it is an essential component in many cellular processes including intracellular membrane trafficking, exocytosis, and apoptosis19,20.
The size of synthesized lipid vesicles often ranges from nanometers to several micrometers. Among different vesicle preparations, giant unilamellar vesicles (GUVs), which are several to tens of micrometers in diameter, are of particular importance due to their relatively large size, closely resembling the dimensions of individual cells21,22,23. The available surface area of the GUVs enables the effect of local chemical gradients on membrane biophysical properties to be studied. By exposing only part of the membrane surface to the external stimuli, the membrane dynamics can be more closely investigated. For example, it has been shown that localized application of chemical or pH gradients to the surface of GUVs leads to the formation of tubular protrusions, which were not observed at bulk exposure24,25. The observed differences in membrane behavior call for further method development of single-vesicle interrogation schemes to gain some insights into the mechanisms of cell membrane remodeling.
Building upon the methods of microinjection and micromanipulation from the early 1900s26,27, in connection with more recent advancements of single-vesicle manipulation schemes from the 2000s23,28, this article presents an approach in which membrane remodeling and formation of membrane tubular protrusions (MTPs) in the GUV membrane are generated in response to local application of calcium ions.
Our approach utilizes a vesicle complex consisting of a GUV connected to a multilamellar vesicle (MLV) as a biomimetic membrane model system (Figure&#160;1A). The MLV is required as a lipid reservoir for the complex to supply lipid material to the GUV during exposure to a calcium ion gradient. This connection allows the complex to compensate for the increase in membrane tension during induced remodeling and shape transitioning of the GUV membrane and provide lipids for MTP growth. Furthermore, the MLV facilitates the surface immobilization because its mass is larger compared to that of the GUV. The GUV-MLV complexes, when immobilized onto a solid substrate, have been previously used for producing nanotube-vesicle networks, studying polymer-membrane interaction, and mimicking the late stages of exocytosis29,30,31,32,33.
Previous protocols utilized soybean polar lipid extract (SPE) to prepare GUV-MLV complexes28. The SPE consists of a mixture of phospholipids that include PC (45.7%), PE (22.1%), phosphatidylinositol (PI, 18.4%), phosphatidic acid (PA, 6.9%), and a mixture of other lipids (6.9%). In our protocol herein, the SPE mixture is doped with 20% of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) to mimic the inner leaflet of the cell plasma membrane. An additional 1% of ATTO 488-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (ATTO488-DOPE) is used to stain the lipid bilayer to enable monitoring of the membrane remodeling using fluorescence microscopy. The GUVs have symmetric lipid composition across the bilayer and are locally exposed to 5 mM concentrations of calcium chloride (CaCl2). Such experimental conditions, with an elevated calcium ion concentration, also mimic the outer membrane leaflet of apoptotic cells, where the PS molecules are expressed34. The formation of the GUV-MLV complexes requires the use of a modified rehydration-dehydration method initially developed by Criado and Keller35. The vesicle preparation protocol includes the formation of a dry lipid layer, which is then used to form small vesicles in solution. This solution is then dehydrated and rehydrated to form the final GUV-MLV complexes. Figure&#160;2A-D illustrates the main steps for the preparation of a typical GUV-MLV complex.
After the vesicle preparation is completed and the vesicle complex is immobilized on the glass substrate, the microinjection technique is used to deliver small amounts of calcium ions to the outer leaflet of the GUV through an open-tip glass micropipette. The flow of calcium solution out of the tip generates a localized calcium ion gradient at the GUV membrane surface, leading to membrane remodeling and generation of the MTPs. The MTPs are oriented away from the calcium ion source and grow inside the GUV. This MTP formation can be directly monitored using fluorescence microscopy and recorded using a digital camera. Figure&#160;3 shows the experimental setup used for producing the membrane remodeling. The formation of the MTPs (Figure 2E and Figure 4) in this protocol demonstrates a contrasting result to calcium ion exposure experiments performed in bulk volume conditions. Under bulk conditions, the GUVs rupture and form membrane patches that can be observed to adhere to the glass surface25.
Further details on the formation of the GUV-MLV complexes, as well as the procedure to perform the microinjection of calcium ions, are explained in this article. The protocols are largely focused on calcium ion microinjection; however, this approach can easily be modified for use in studying the membrane responses due to local exposure to other ions or proteins. Additionally, the composition of the vesicles can be tuned to isolate the lipid component roles in the process of membrane remodeling. The presented protocol does not require any sophisticated equipment to produce the GUV-MLV complexes and is characterized by a high degree of reproducibility.

<table><tbody><tr><td>Soy bean polar lipid extract</td><td>Avanti&lt;br/&gt; Polar Lipids, Inc. (Alabaster, USA)</td><td>541602C</td><td>100 mg</td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) DOPS</td><td>Avanti&lt;br/&gt; Polar Lipids, Inc. (Alabaster, USA)</td><td>840035C</td><td>1x25 mg</td></tr><tr><td>ATTO 488- 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)</td><td>ATTO-TEC (Germany)</td><td>AD 488-31</td><td>1 mg</td></tr><tr><td>Hamilton&amp;nbsp;syringe, 700 series, fixed needle, 702N, volume 25&amp;nbsp;&amp;mu;L, needle size 22s ga (bevel tip), needle L 51&amp;nbsp;mm (2&amp;nbsp;in.)</td><td>Sigma Aldrich (Missouri, USA)</td><td>20735&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Pyrex Tube, culture, disposable, rimless, 10x75 mm, Borosilicate glass 250/pack</td><td>Corning Incorporated (Corning, NY 14831)</td><td>99445-10</td><td></td></tr><tr><td>Chloroform&amp;nbsp;CHROMASOLV&amp;nbsp;Plus, for HPLC, &amp;ge;99.9%, contains amylenes as stabilizer</td><td>Sigma Aldrich (Missouri, USA)</td><td>650498-1L-D</td><td></td></tr><tr><td>Rotary evaporator</td><td>B&amp;uuml;chi Rotavapor R-144 Switzerland</td><td></td><td></td></tr><tr><td>Kalciumklorid purum torkad minimum 95% medelkornig 5-10 mm</td><td>KEBO lab (Sweden)</td><td>MA00360500</td><td></td></tr><tr><td>Magnesium chloride hexahydrate reagent grade ACS, ISO</td><td>Sharlau Chemie S.A. (Spain)</td><td>P9333-500G</td><td></td></tr><tr><td>Potassium chloride, SigmaUltra, minimum 99.0%</td><td>Sigma Aldrich (Missouri, USA)</td><td>S7653-1KG</td><td></td></tr><tr><td>Sodium chloride, SigmaUltra, minimum 99.5%</td><td>Sigma Aldrich (Missouri, USA)</td><td>G5516-1L</td><td></td></tr><tr><td>Glycerol, for molecular biology, minimum 99%</td><td>Sigma Aldrich (Missouri, USA)</td><td>T-1503 2050 g</td><td></td></tr><tr><td>Trizma base</td><td>Sigma Aldrich (Missouri, USA)</td><td>P5629-500G</td><td></td></tr><tr><td>Potassium phosphate tribasic (K3PO4)</td><td>Sigma Aldrich (Missouri, USA)</td><td>P5655</td><td></td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Sigma Aldrich (Missouri, USA)</td><td>5886</td><td></td></tr><tr><td>MgSO4</td><td>Merck (USA)</td><td>34549-100 g</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich (Missouri, USA)</td><td>H0887 Sigma&amp;nbsp;</td><td></td></tr><tr><td>HEPES solution 1&amp;nbsp;M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture</td><td>Sigma Aldrich (Missouri, USA)</td><td>Z260282-1PAK</td><td>24x60 mm</td></tr><tr><td>Acrodisc&amp;nbsp;syringe filters,&amp;nbsp;PVDF membrane, diam. 13&amp;nbsp;mm, pore size 0.2&amp;nbsp;&amp;mu;m</td><td>Sigma Aldrich (Missouri, USA)</td><td>631-1339&amp;nbsp;</td><td></td></tr><tr><td>Menzel Gl&amp;auml;zer #1, glass cover slip</td><td>VWR (USA)</td><td></td><td></td></tr><tr><td>Diaphragm vacuum pump for the desiccator</td><td>Vacuubrand (Germany)</td><td></td><td></td></tr><tr><td>Ultrasonicate bath</td><td>Bandelin Sonolex (Germany)</td><td></td><td></td></tr><tr><td>VX-100 Lab vortexer vortex mixer</td><td>Labnet International (USA)</td><td></td><td></td></tr><tr><td>488 nm laser line&amp;nbsp;</td><td>Cobolt MLD-488 nm (Solna, Sweden)</td><td></td><td></td></tr><tr><td>Leica Microsystems&amp;nbsp;immersion oil for microscopes</td><td>Leica (Germany)</td><td>12847995&amp;nbsp;</td><td></td></tr><tr><td>Inverted fluorescence microscopy system&amp;nbsp;</td><td>Leica DM IRB (Wetzlar, Germany)</td><td></td><td></td></tr><tr><td>Camera (Prosilica Ex 1920, Allied Vision)</td><td>Technologies GmbH (Thuringia, Germany)</td><td>300038</td><td></td></tr><tr><td>PatchStar Micromanipulator</td><td>Scientifica (Uckfield, UK)</td><td>612-7933</td><td></td></tr><tr><td>Borosilicate glass capillaries, GC100TF-10,&amp;nbsp; 1.00mm O.D. X 0.78mm I.D.&amp;nbsp;</td><td>Harvard Apparatus U.K</td><td></td><td></td></tr><tr><td>Eppendorf microloader (pipette tips)</td><td>VWR (USA)</td><td></td><td></td></tr><tr><td>P-2000 CO2 laser-puller&amp;nbsp;</td><td>Sutter Instruments (Novato, USA)</td><td></td><td></td></tr><tr><td>Femtoliter automatic injection pump, Eppendorf Femtojet</td><td>Eppendorf (Germany)</td><td></td><td></td></tr></tbody></table>

membrane remodeling of giant vesicles in response to localized calcium ion gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

In this work, we demonstrate the creation of GUV-MLV complexes and how they can be used to elucidate the effect of calcium ion gradients on cell membrane dynamics. Surface-attached GUV-MLV complexes are required for these experiments to allow for a fixed cell-sized mimic while establishing an ion gradient through microinjection. The GUV membrane responds to localized calcium concentration through formation of MTPs directed away from the calcium ion source. Furthermore, the MTPs can be translated around the GUV in a contactless manner by moving the micropipette tip around the membrane surface.
A schematic illustration of the GUV-MLV preparation procedure is shown in Figure&#160;2. Although in the presented protocol, the GUV-MLV complexes are already pre-formed during step 2.4 (Figure&#160;2C), transferring the vesicle solution to another 300 &#181;L droplet of buffer (step 2.6 of the protocol) allows the sparsely formed GUV-MLV complexes to sufficiently adhere to the glass substrate. In this way, a suitable complex for micromanipulation and microinjection is established (Figure&#160;2D). Occasionally, the GUVs contain one or several entrapped lipid vesicles, which do not affect the MTP formation, as shown in Figure&#160;4A. These GUVs produce MTPs; however, the imaging may be obstructed if the entrapped vesicles are large. The majority of the prepared GUVs (up to 75%) appear hemispherical and are attached to the MLVs, as shown in Figure 1A. These vesicles are the main focus of this protocol and are suitable for the microinjection procedure. About 25% of the remaining GUVs appear undulating (Figure&#160;1B), which is indicative of a lower membrane tension regime. These undulating vesicles also allow for the formation of the tubular protrusions; however, they exhibit different kinetics and a different morphology of the formed MTPs, which is beyond the scope of this protocol25. The typical diameter of the prepared vesicles varies between 2 to 15 &#181;m for the MLVs, and between 2 to 40 &#181;m for the GUVs. The optimal GUV size for the exposure to calcium ions is 5 &#181;m or larger.
Micropipettes have been used in singe-cell interrogation schemes as well as in procedures requiring manipulation of synthetic GUVs for several decades37,38. The majority of these applications involved direct physical contact between the micropipette and the surface of cells or vesicles. Examples include the patch-clamp technique or pulling membrane tethers from the GUV membrane, in addition to building nanotube-vesicle networks23,28,39. Recently, micropipettes have also been used to generate localized gradients of ions and molecules around GUVs to reconstruct heterogeneous cell microenvironments24,25. In the presented protocol, a properly functioning micropipette (Figure&#160;5A) is a crucial factor for establishing a calcium ion gradient at the GUV surface by releasing the solution contained within. Proper positioning of the micropipette at the GUV surface and avoiding contact with the lipid membrane are essential for successful microinjection. The micropipette tip is held at a distance of approximately 3 &#181;m from the GUV surface, which is an optimal distance to generate MTPs while mimicking variations of calcium near the cell membrane. If the micropipette tip is accidently broken (Figure&#160;5B), a replacement is required. The previously tested concentration range of CaCl2 inside the micropipette, which allows for the MTP formation, is between 2 and 5 mM25, which corresponds to extracellular calcium concentrations. At lower CaCl2 concentrations, i.e., 1 mM, no MTPs are observed.
The formation of MTPs upon the calcium microinjection begins with the formation of small membrane invaginations, which grow into MTPs at the site of the exposure (Figure&#160;4B). The MTPs continue to grow as long as the GUV membrane remains exposed to the calcium ions. They fluctuate and point away from the calcium ion source (Figure 4C, Supplementary Movie 1). When the calcium ion supply is terminated, the MTPs scatter over the GUV surface, making it difficult to conclude whether the MTPs remain or eventually disappear.
The formation of MTPs can be explained by localized calcium ion binding to the exposed outer leaflet of the GUV membrane and triggering spontaneous curvature (m), which is directly associated with the formation of spontaneous tension &#963; = 2&#954;m2 (&#954; is the bending rigidity), that induce bending of the membrane and forming inward MTPs. Previous reports have demonstrated that deformation of the membrane can be explained by condensation and/or clustering of the negatively charged lipids upon binding of the calcium ions to the membrane, resulting in negative spontaneous curvature40. Alternatively, the strong binding of Ca2+ to the surface of the negatively charged bilayer neutralizes the surface charge density of the exposed lipid bilayer leaflet. This leads to the charge density difference across the bilayer, resulting in nonzero spontaneous curvature, sufficient to bend membrane25.
The observed formation of MTPs demonstrates a sensitivity of lipid membranes to calcium ions and offers a novel contactless approach for producing membrane tubular structures. Elucidating the origin of this membrane tubulation can be important for understanding the dynamics of membrane shape transitioning during various cell functions and cell reshaping and can be helpful for gaining insights into lipid organization within cell membranes.
The observed MTPs are always generated at the site on the membrane undergoing calcium ion exposure. Thus, by choosing the location of the micropipette tip, it is possible to define the area of the GUV surface for the protrusion growth. Next, if the micropipette is slowly (0.1-0.3 &#181;m/s) moved around the GUV surface, while maintaining the separation distance from the membrane, the MTPs are translated in tandem with the micropipette. Figure&#160;6 and the corresponding Supplementary&#160;Movie 2 demonstrate such migration of the MTPs around the GUV surface. This process is likely to be accompanied by the formation of new MTPs upon continuous calcium ion injection25. At high translation rates (above 0.7 &#181;m/s), the MTPs are not able to follow the micropipette tip. Instead, new protrusions are formed at the new location of the micropipette tip (Figure&#160;7).
The observed contactless calcium ion-guided translation of MTPs around the GUV surface can further our understanding of the driving forces behind the dynamics of membrane tubular structures inside cells. Moreover, this approach offers a novel contactless mode for controlling the transport of material in soft matter systems by using chemical gradients.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57789/57789fig1.jpg" /> 
Figure 1: Representative fluorescent microscopy images of GUV-MLV complexes immobilized on the surface of a glass cover slip. (A). Example of a spherical GUV. The GUV appears in the form of a hemisphere attached to the MLV. (B). Example of an undulating GUV. The black arrows indicate the deformed areas of the membrane. The images are enhanced and inverted to improve the visualization of the fluorescently labeled GUV membranes. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/57789fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57789/57789fig2.jpg" /> 
Figure 2: Schematic illustrations of the GUV-MLV preparation and the microinjection schemes. (A). A dry lipid layer is formed at the bottom of the glass vial as a result of the rotary evaporation of chloroform from the lipid solution. (B). The rehydrated lipid layer is sonicated, and small lipid vesicles are formed. (C). A small droplet of the vesicle solution (5 &#181;L) is placed on the surface of a glass cover slip and transferred into a desiccator for 20 min to dehydrate the lipid solution and form a dry lipid film (this step is not shown). Rehydrating the dry lipid film with 50 &#181;L of buffer solution produces the pre-formed GUV-MLV complexes. (D). The transfer of the pre-formed complexes to a larger volume of buffer results in the formation of sparsely separated GUV-MLVs attached to the surface of the glass cover slip. (E). Positioning the micropipette near the surface of the GUV and releasing the calcium ions triggers the formation of MTPs. The illustrations are not drawn to scale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/57789fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57789/57789fig3.jpg" /> 
Figure 3: Experimental setup for the microinjection of calcium ions. (A). The components of the experimental setup required to generate the MTPs in the GUVs. (B). Details of the microinjection setup. The glass coverslip with the solution of the GUV-MLV complexes placed on the microscope stage. The angle between the micropipette and the surface of the glass cover slip is 30&#176; (marked in white). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/57789fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57789/57789fig4.jpg" /> 
Figure 4: Formation of MTPs upon the microinjection of the calcium ions at the surface of the GUV. (A). The GUV-MLV complex prior to exposure to a calcium ion gradient. The arrow indicates a lipid vesicle entrapped inside the GUV, and which does not obstruct the observation of the MTPs. (B). Small MTPs are formed upon the microinjection of calcium ions (5 mM concentration of CaCl2 in the micropipette). (C). Growth of the MTPs upon continuous exposure to calcium ions. The fluorescence images are enhanced and inverted for visualization purposes. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/57789fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57789/57789fig5.jpg" /> 
Figure 5: Glass micropipettes used for calcium ion microinjection. (A). An example of a properly functioning micropipette. (B). An example of a micropipette that has a broken tip (the damaged area is indicated with a black arrow). Both images are enhanced and inverted for better visualization. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/57789fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57789/57789fig6.jpg" /> 
Figure 6: Calcium ion-guided translation of the MTPs around the surface of the GUV. (A). The MTPs are formed at the GUV surface upon microinjection of 5 mM CaCl2 solution. (B-C). Translation of the micropipette tip (0.2-0.3 &#956;m/s) around the membrane surface triggers the movement of the MTPs in the direction of the calcium ion source. The black arrows highlight the direction of the micropipette movement. The images are enhanced and inverted to improve membrane visualization. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/57789fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57789/57789fig7.jpg" /> 
Figure 7: MTPs at high translation rate of the micropipette tip. (A). The MTPs are formed at the GUV surface upon the microinjection of the 5 mM CaCl2 solution (white line). (B-C). A high translation rate of the micropipette around the GUV surface (1 &#956;m/s) results in the formation of new MTPs at the new location of the micropipette tip (magenta line), as well as scattering of the previously formed MTPs (white line). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/57789fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/57789/57789mov1.jpg" /> 
Supplementary Movie 1: Formation of MTPs in GUV upon localized exposure to the Ca2+ gradient.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/Lobovkina_Movie_1.avi" target="_blank">Please click here to download this movie.</a>
<img alt="Movie 2" src="/files/ftp_upload/57789/57789mov2.jpg" /> 
Supplementary Movie 2: Calcium ion-guided MTP migration around the GUV surface.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57789/Lobovkina_Movie_2.avi" target="_blank">Please click here to download this movie.</a>

Watch this Scientific Journal Video about Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients at JoVE.com

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

membrane-remodeling-giant-vesicles-response-to-localized-calcium-ion

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.9K Views.  Chalmers University of Technology. Presenteren we een techniek voor contactloze micromanipulation van blaasjes, met behulp van de gelokaliseerde calcium ion verlopen. Microinjection van een calcium Ionenoplossing, in de nabijheid van een gigantische lipide blaasje, is gebruikt om het remodelleren van het lipide membraan, wat resulteert in de productie van membraan buisvormige uitsteeksels.

Video: Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

Biochemistry

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Biology

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Bioengineering

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Neuroscience

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Pinching-off of Coated Vesicles

Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming the coated vesicle.
COP-coated vesicles bud off their organelle membrane with the help of several small GTPases such as SEC, SAR, and ARF proteins. On the other hand, Clathrin-coated vesicles require additional proteins, the most important of which is Dynamin. Dynamin is a GTPase of about 100-kDa that assembles as helical spirals at the necks of vesicle buds. It undergoes a GTP hydrolysis-driven conformational change that results in a twisting motion, which detaches the clathrin-coated vesicle from the membrane.
Once a vesicle buds, it immediately sheds its coat. The energy required for uncoating the clathrin coat is derived through ATP hydrolysis by Hsp70 chaperone protein, an ATPase activated by a co-chaperone named auxin. Auxilin binds Hsp70 and guides it to appropriate locations on the clathrin lattice to stimulate ATP hydrolysis. Auxilin binding creates a detectable distortion in the clathrin coat. It simultaneously contacts another clathrin at its terminal domain, recruiting more Hsc70 to the neighborhood, thus uncoating the entire vesicle. Tight control of vesicular coat formation and shedding facilitates the timing of vesicle budding and fusion with the target membrane.

Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, ...

Education

JoVE Core

Cell Biology

Unit 1

Intracellular Membrane Traffic

KEY TERMS AND CONCEPTS

Fusion of Secretory Vesicles with the Plasma Membrane

Proteins and neurotransmitters in secretory vesicles can be released from a cell upon vesicle docking, priming, and fusion with the plasma membrane. Vesicles are docked and primed in preparation for the quick exocytosis of their contents in response to a stimulus. The fusion process is mainly carried out by a SNAP Receptor or SNARE complex, consisting of synaptobrevin, syntaxin-1, and SNAP-25.
In 1993, Jim Rothman proposed that the antiparallel pairing of vesicular and transmembrane SNAREs, or v- and t-SNAREs, was essential for vesicle docking. However, more recently, it has been shown that vesicle docking can also occur without SNAREs. Additionally, two soluble proteins,&#160; N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP), bind to the SNARE complex to facilitate fusion.
The v-and t-SNAREs partially fuse in the priming process, forming a fusion-ready state. The protein complexin (Cpx) clamps the SNAREs and holds the vesicles in this partially fused state to prevent premature exocytosis. Vesicular membrane fusion begins when a stimulus, such as an action potential at the axonal terminal of a neuron, opens up a calcium channel, and calcium enters the cell. A total of five calcium ions bind to each synaptotagmin (Syt) &#8211; a vesicular membrane protein on either side of the vesicle. Calcium-bound Syt releases the Cpx clamp from the SNARE complexes and opens up the fusion pore to release the neurotransmitter. After fusion, NSF and SNAP proteins disassemble SNARE complexes for recycling.
Bacterial neurotoxins, such as botulinum from Clostridium botulinum or tetanus from Clostridium tetani, can inhibit secretory vesicle fusion by damaging the SNARE proteins, which prevents the fusion of secretory vesicles with the neuronal plasma membrane. As neurotransmitters are not released, action potentials are not generated, causing paralysis of muscles, and in some cases, death.

Proteins and neurotransmitters in secretory vesicles can be released from a cell upon vesicle docking, priming, and fusion with the plasma membrane. ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

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